Tuberculosis 1: Mycobacteria And Diagnosis Of Mycobacterial Infections Flashcards
1
Q
Mycobacterium
A
- ***Gram +ve Bacilli
- ***Obligate aerobe
- Slow growth rate
- High lipid content of cell wall —> ↓ permeability to antibiotics + staining properties
- Acid-fast staining —> Ziehl-Neelsen stain
- Unique antibiotic susceptibility patterns
2
Q
Classification of mycobacteria
A
- Mycobacterium tuberculosis complex
- M. tuberculosis (commonest)
- M. bovis
- M. bovis BCG (bacillus Calmette-Guerin) - Mycobacterium leprae
- uncultivable with routine technique
- diagnosis: Clinical, Histopathology, Nucleic acid amplification - Other Mycobacteria (Non-tuberculous mycobacteria NTM / Mycobacteria other than tubercle bacillus MOTT / Atypical mycobacteria)
- Runyon classification previously based on **growth rates + formation of **pigments —> Definitive identification: Molecular techniques
- Runyon group 1: **Photochromogens —> produce pigments in light (e.g. M. kansaii)
- Runyon group 2: **Scotochromogens —> produce pigments in / without light (e.g. M. scrofulaceum)
- Runyon group 3: **Non-chromogens —> do not produce pigments (e.g. M. avium complex)
- Runyon group 4: **Rapid growers —> visible growth within 7 days after subculture (e.g. M. fortuitum complex)
3
Q
Diseases caused by mycobacteria
A
- Very diverse
- Opportunistic
M. tuberculosis:
- ***Pulmonary + Extrapulmonary TB
- Primary + Post-primary TB
M. leprae:
- ***Leprosy
NTM:
- Pulmonary infections: M. kansasii
- Lymphadenitis: M. scrofulaceum
- ***Skin + soft tissue infections: M. marinum
- Disseminated infection in immunocompromised: M. haemophilum
- Catheter-related infections: Rapid growers
- ***Nosocomial infection (e.g. contaminated devices, post-injection abscess, inadequate sterilisation of equipment): Rapid growers
- Outbreaks associated with heater-cooler units in cardiac surgery: M. chimaera
4
Q
Clinical specimen
A
M. tuberculosis: ***Always significant
Other mycobacteria: Significant if in sterile sites e.g. blood, tissue
NTM: maybe Colonisation if in sputum / superficial wound swabs
—> Need to correlate with **clinical + **radiological findings
5
Q
Laboratory identification of Mycobacteria
A
- Conventional: Physiological + Biochemical tests
- MALDI-TOF MS: Rapid identification possible but accuracy depends on quality of MS database
- ***Molecular techniques (PCR + Sequencing): Most reliable
- Positive bacterial culture
- **gold standard in microbiological diagnosis in most cases
- **very sensitive
- allow exact species identification + antibiotic susceptibility testing
—> antibiotic treatment very different for different species esp. NTM
- allow antibiotic resistance detection esp. M. tb
- allow epidemiological typing in outbreak investigations
- ***however, slow growth rates + low bacterial load
6
Q
***Drug resistance M. tuberculosis
A
- MDR-TB
- Isoniazid + Rifampicin - XDR-TB
- MDR + any Fluoroquinolone + >=1 of 3 injectable second-line drugs (Amikacin, Kanamycin, Capreomycin) - TDR-TB
- All 1st + 2nd line drugs
7
Q
Antimicrobial susceptibility testing
A
- M. tuberculosis
- **Phenotypic method: culture isolate in presence of antimicrobial agents —> look for inhibition of growth
- **Genotypic assay: detect specific genes but resistance can be mediated by different gene mutations / multiple mechanisms which are not detected - M. leprae
- Routine testing not possible - NTM
- no standardised testing method
8
Q
***Laboratory diagnostics of Mycobacterial infections
A
- ***Clinical suspicion
- ***Radiology
- ***Acid-fast stain
- ***Culture
- Tuberculostearic acid (TBSA) (not commonly used now)
- Adenosine deaminase (ADA)
- Urine lipoarabinomannan (LAM) antigen
- Antibody detection
- Tuberculin skin test
- In vitro interferon-γ release assay (IGRA)
- ***Nucleic acid amplification
Empirical treatment against TB may be given even infection not definitively confirmed by laboratory investigations, based on
- clinical picture
- suggestive epidemiological, radiological, pathological, other laboratory findings
NTM: Positive bacterial culture / PCR
9
Q
Clinical specimens for diagnosis of mycobacterial infections
A
Quality + Quantity (e.g. volume of CSF)
Respiratory specimens:
- ***Sputum
- Lower respiratory tract: ***Bronchoalveolar lavage +/- Transbronchial biopsy / Transthoracic needle biopsy of lung
- when lack of sputum / respiratory symptoms
- failure of less invasive procedures
- certain forms of TB e.g. Endobronchial TB
- exclusion of concurrent infections / non-infectious pathologies e.g. malignancy
10
Q
Diagnosis of extrapulmonary TB
A
AFB smear: ***Low sensitivity
- Tissue biopsy at infection sites
- Nucleic acid amplification tests (not necessarily high sensitivity)
- Histopathology
11
Q
Clinical syndromes of TB
A
- Epidemiological context e.g. age, contact
- Radiological features
- NOT distinguish TB from NTM
- pulmonary TB not limited to lung apices
- chest radiograph can be ***normal in endobronchial TB - Underlying conditions
- immunocompromised
- HIV
- treatment with biologics - Other relevant lab findings
- ***CSF cell counts + biochemistry in patients with meningitis - Latent vs Active TB
12
Q
***Latent vs Active TB
A
Latent TB (LTBI):
- ***Negative bacteriological diagnosis
- ***Suspicion from radiological findings
- diagnosis: **Immunological tests e.g. **tuberculin skin test, ***IGRA
- treatment: ***1 / 2 drug regimen to ↓ recurrence risk
Active TB:
- ***Positive (more likely) bacteriological diagnosis
- Immunological tests cannot differentiate active vs latent
- treatment: ***standard 4 drug regimen
13
Q
Microscopy / Smear
A
- Simple, inexpensive, rapid
- Operator-dependent
- ***Low sensitivity: require >=10^4 organisms per ml of sputum, many patients have negative smear
- cannot differentiate M. Tb from ***NTM by morphology alone
- cannot differentiate living vs ***dead AFB using conventional acid-fast stain
- ***quick guide to infectiousness of pulmonary TB: smear positive vs negative + infection control implications
14
Q
Mycobacterial culture
A
Lager volumes of specimen better e.g. min 5ml of CSF
15
Q
Serology
A
No use
16
Q
Adenosine deaminase (ADA)
A
- Derived from ***host lymphocytes + monocytes
- ***not specific to M. Tb and its infection
- used in pleural, pericardial, CSF as adjunct to standard bacteriological testing
17
Q
Urine lipoarabinomannan (LAM) antigen detection
A
- Major ***glycolipid cell wall component of M. Tb
- Inexpensive
- done using ***ELISA / point-of-care testing using immunochromatographic format
- high sensitivity in ***HIV patients with CD4 count <200/ml
- poor sensitivity in non-HIV patients
18
Q
***Nucleic acid amplification tests e.g. PCR
A
Advantages:
- Sensitivity: depends on bacterial load + type of specimens
- Specificity 95-100%
- ***Fast turnaround time
- ***Species identification possible
- Rapid detection of ***resistance genes
- Fully automated in some commercial NAAT systems
Disadvantages:
- Cost
- ***Limited sensitivity for paucibacillary disease + extrapulmonary TB
- Inhibitors in specimens may affect test
- No definitive information on drug susceptibility / resistance
- ***Only supplement but not replace conventional bacteriological testing
19
Q
Tuberculin skin test / Mantoux method
A
Procedure:
- Intradermal injection of 0.1ml ***purified protein derivative (PPD)
- RT-23 PPD: each 0.1ml consists of ***2 TU (tuberculin units)
- wheal 6-10mm in diameter should be produced
- inject another at once at a different site if 1st dose not administered correctly
- record site, date, time of administration in medical record
Delayed-type hypersensitivity response:
- person exposed to bacteria before will mount ***immune response in skin to bacterial proteins
- read results at ***48-72 hours
- record maximal transverse diameter (mm) of ***induration but not erythema
- measured transversely to long axis of forearm at widest diameter
- 0mm for absence of induration (X negative)
- not equivalent to protective cell-mediated immunity against M. Tb
False negative:
- Technical (e.g. inadequate dose)
- Administration
- Reading (e.g. biased reader)
- Biological (e.g. viral infections, HIV, measles, disseminated TB, malignancy)
False positive:
- ***BCG vaccination
- ***NTM exposure
Cut-off value:
- ***5mm: HIV-infected individual / immunocompromised patients
- ***10mm: most individuals
20
Q
In vitro interferon-γ release assay (IGRA)
A
Alternative to TST
2 test formats:
1. QuantiFERON-TB Gold
2. T-SPOT TB
—> both uses peripheral blood for testing
Principle:
- stimulate T cells of individual using mycobacterial antigens in vitro
- release of **IFN-γ by T cells in response to **MTB-specific antigens
- mononuclear cells from MTB-infected people will have higher level of IFN-γ production upon exposure to mycobacterial antigens
Antigens used:
- Early secretory antigenic target 6 (ESAT6)
- Culture filtrate protein 10 (CFP10)
Advantages:
- Both higher ***specificity than PPD as not being produced by BCG strain and most NTM
- Less affected by previous BCG vaccination / NTM infections
Disadvantages:
- Neither TST / IGRA distinguish ***latent vs active TB
- Also affected by underlying ***immune status e.g. HIV
21
Q
Histopathology + Cytology
A
Advantages:
- Useful when bacilli are few, yet still detectable host responses
- If very typical pathology seen (e.g. Langhans giant cells, caseous necrosis) —> suspect most likely causative agent
Disadvantages:
- Even if AFB present —> morphological findings does not definitely identify the exact species
- Host response can be modified by immune status (e.g. HIV: suppurative response rather than granulomatous inflammation)
- No information on drug resistance
